Abstract
Abstract: :
Purpose: Lumican is a keratan sulfate proteoglycan of the corneal stroma secreted by stromal keratocytes. In healing stromal wounds, activated keratocytes secrete a glycoprotein form of lumican without keratan sulfate chains. We recently reported (J Biol Chem 275, 2607) that after wounding, corneal epithelial cells transiently express lumican mRNA and protein. Anti–lumican antibody reduced epithelial cell migration rate and lumican knockout mice exhibit reduced epithelial wound healing, restored by addition of soluble lumican. The role of lumican in epithelial migration suggests the possibility of similar function in stromal healing. This study examined lumican as a mediator of keratocyte motility. Methods: Primary bovine keratocytes were grown to confluence in 10% serum–containing medium then maintained serum–free. The cells were fluorescently labeled with Di–O lipophilic dye, trypsinized and plated on FluoroBlok transwell membrane filters with 8 µm pores in serum–free DME/F12. The membranes were pre–coated with bovine serum albumin, glycoprotein lumican from aorta, keratanase–treated corneal lumican, collagen type I, or fibronectin. The lower chamber contained DME/F12 with 10 ng/ml fibroblast growth factor 2 (FGF2). Membranes were photographed with an inverted fluorescent microscope at 2 hr, 6 hr and 24 hr after plating. Cell numbers in five fields were averaged for each culture condition and time point. Results: Primary stromal fibroblasts migrate rapidly in response to FGF2 when plated on collagen or fibronectin but exhibit little migration on filters coated with albumin. The glycoprotein form of lumican isolated from artery, as well as lumican from cornea after enzymatic removal of keratan sulfate chains, stimulated keratocyte migration. Response to lumican was significantly greater than that to fibronectin but less than the stimulation by collagen. Affinity purified antibody to corneal lumican inhibited the migration on lumican. Conclusions: As with corneal epithelial cells, the glycoprotein form of lumican supports migration of stromal cells. The secretion of a glycoprotein form of lumican by activated keratocytes may enhance the migration of these cells during wound repair
Keywords: extracellular matrix • cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans